GPR68 is an understudied orphan G-protein coupled receptor (GPCR). It can sense extracellular pH changes, therefore functioning as a H+-sensing receptor. It couples to multiple signaling pathways and is implicated in many biological processes, including pH homeostasis, inflammation, and vascular physiology. It is expressed in most cells, but most abundantly in the brain, especially hippocampus ? the brain region that is critical to learning and memory and can also be affected in brain or mental disorders with cognitive deficits. Underlying molecular mechanisms of GPR68 pharmacology and biology are not clear and yet to be defined. Pharmacological studies with GPR68 have been hindered by the lack of small molecule tool compounds that can be used to selectively activate or inhibit its activity. Selective tool compounds for GPR68 are urgently needed. In 2015, we discovered the first small molecular ligand, ogerin, a positive allosteric modulator (PAM) of GPR68, providing the first tool compound to selectively activate GPR68 (Huang et al., Nature 2015). This selective probe led to the discovery that GPR68 suppresses contextual- but not cue-dependent learning and memory in wild-type but not GPR68 knock-out mice, the first evidence indicating a role for GPR68 in cognition. This observation suggests a potential link between GPR68 activation and cognitive deficits associated with brain injuries and certain brain disorders, such as schizophrenia and neurodegenerative disease; these conditions are usually associated with inflammation and brain regional acidosis (reduced pH), which could activate GPR68. To study GPR68 pharmacology and to demonstrate if blocking GPR68 activation can help learning and memory or reduce cognitive deficits, especially under weak acid conditions, selective GPR68 antagonists are needed. A current gap in our ability to exploit these possibilities is the lack of a small molecule antagonist for GPR68, that can selectively block its activity in pharmacological assays. Our recent discovery of the first selective GPR68 PAM-antagonist, MS27101, is a crucial step forward towards discovering such a selective inhibitor, and is the focus of this application. Allosteric modulators such as ogerin bind to GPR68 at a site different from that of H+, and fine tune H+ activity through increasing or reducing H+ affinity (?- cooperativity), efficacy (?-cooperativity), or both. In contrast to traditional orthosteric agonists or antagonists which rely on binding affinity for selectivity, allosteric modulators achieve high selectivity through allosteric ?? cooperativity in addition to binding affinity (KB). PAM-antagonists are a unique type of negative allosteric modulator (NAM) with affinity cooperativity ?>1 and efficacy cooperativity ?<1. Specifically, MS27101 affinity (KB) is enhanced by H+ (because of ?>1), locking it into its binding pocket to block H+ activity (because of ?<1) only under acidic conditions, while it remains inactive under normal pH conditions. Since GPR68 can have pathological activity at lower pH levels, the selective antagonism at low pH provides a uniquely beneficial therapeutic profile. GPR68 is always exposed to H+, and the histidine binding sites for H+ are difficult to directly block, making allosteric modulation attractive. MS27101 has high selectivity over related H+-sensing GPCRs (such as GPR4 and GPR65), but has relatively low affinity and efficacy at GPR68. One approach to design and develop novel allosteric modulators is to modify an existing one. In this proposal, with MS27101 as a starting scaffold, we will carry out extensive structure-activity relationship (SAR) studies to modify MS27101 structure to design potent (?>1 and increased affinity KB) and efficacious (?<1) PAM-antagonists to block acid- associated GPR68 activity. Selected PAM-antagonists, together with PAM ogerin, can be used in combination to manipulate (activate or inhibit) GPR68 activity and advance GPR68 pharmacology and biology studies with possible relevance to new therapeutic entities.

Public Health Relevance

GPR68 is an understudied orphan G-protein coupled receptor (GPCR) and capable of sensing extracellular proton concentrations. It has been implicated in many biological processes, but GPR68 pharmacological studies have been hindered by the lack of small molecule compound that can be used to selectively inhibit its activity. We recently identified the first GPR68 antagonist and we propose to design and develop more potent and efficacious antagonists that can be used as tool compounds to selectively block GPR68 activity under weak acidic conditions.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Exploratory/Developmental Grants (R21)
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Drug Discovery for the Nervous System Study Section (DDNS)
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Michelotti, Enrique
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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